One of the largest families of receptors in the human genome is that of the 7 transmembrane receptor (7-TMR) superfamily, also known as G-protein coupled receptors (GPCRs), numbering approximately 2000 proteins. G-protein coupled receptors regulate a large number of important physiological processes. At least 40% of the prescription drugs that have been developed have actions related to these receptors. Most of these drugs work by interfering with the ligand binding to the receptor that occurs outside of cells.
There is an ongoing effort in the scientific community to define compounds that block the intracellular interaction between the receptor and its signal transducing partner, the G-protein.
The second intracellular loop of the 7TMR receptors is known to play an important role in the signal transduction as mutations in this region cause a disturbance in the 7TMR-associated signal transduction. For example, there has been an attempt (Benovic et al., Br. J. Clin. Pharmac., 30:3s-12s, 1990) to interfere with the β2-adrenoreceptor signal transduction by administration of peptides corresponding to the full second loop of this receptor. However, these results were extremely unsatisfactory.
Angiogenesis is an important natural process that occurs during embryogenesis, and in the adult healthy body in the process of wound healing, and results in restoration of blood flow back into injured tissues. In females, angiogenesis also occurs during the monthly reproductive cycle to build up the uterus lining and to support maturation of oocytes during ovulation, and in pregnancy when the placenta is formed, in the process of the establishment of circulation between the mother and the fetus. The healthy body controls angiogenesis through the interactions of angiogenesis-stimulating growth factors, and angiogenesis inhibitors, and the balance between the two determines whether angiogenesis is stimulated or inhibited.
In several therapeutic fields, there has been a growing interest in the control of angiogenesis. In one aspect, the aim is to control or diminish excessive and pathological angiogenesis that occurs in diseases such as cancer, diabetic blindness, age related macular degeneration, rheumatoid arthritis, psoriasis, and additional conditions. In these pathological conditions the new blood vessels feed the diseased tissue, for example the tumor tissue, providing it with essential oxygen and nutrients thus enabling its pathological growth. In some conditions the pathological angiogenesis many times destroys the normal tissue. Furthermore, the new blood vessels, formed for example in the tumor tissue, enable the tumor cells to escape into the circulation and metastasize in other organs. Typically, excessive angiogenesis occurs when diseased cells produce abnormal amounts of angiogenetic growth factors, overwhelming the effect of the natural angiogenesis inhibitors present in the body.
Anti-angiogenic therapies developed today are aimed at preventing new blood vessel growth through the targeting and neutralization of any of the stimulators that encourage the formation of new blood vessels.
Another aim of regulating angiogenesis is the stimulation of production of neovascularization in conditions were insufficient blood-supply occurs, leading to tissue ischemia. Typically, these conditions are diseases such as coronary artery diseases, peripheral artery diseases, stroke, and delayed wound healing (for example in ulcer lesions). In these conditions, when adequate blood vessel growth and circulation is not properly restored, there is a risk of tissue death due to insufficient blood flow. Typically, insufficient blood-supply occurs when the tissues do not produce adequate amounts of angiogenic growth-factors, and therapeutic angiogenesis is aimed at stimulating new blood vessels' growth by the use of growth factors or their mimics.
The main goal of the angiogenesis promoting therapy is to produce a biobypass—i.e. to physically bypass diseased or blocked arteries, by tricking the body into building new collateral blood vessels.
Sphingosine 1-phosphate (S1P) has been shown to be a major pro-angiogenic factor in the serum (English et al., Cardiovasc Res. 2001; 49:588-599). S1P is released from platelets on their activation and contributes to wound healing processes. Its production is catalyzed by sphingosine kinase, while degradation is either via cleavage to produce palmitaldehyde and phosphoethanolamine or by dephosphorylation. S1P serves as a key element in the angiogenic response by triggering proliferation and migration of endothelial cells as well as capillary morphogenesis, and stabilizing the new-formed endothelial capillary, by enhancing cell-cell contacts and recruiting smooth muscle cells. S1P can also bind to members of the endothelial differentiation gene (EDG) G-protein-coupled receptor family (namely EDG1 also known as S1P1, EDG3 also known as S1P3, EDG5 also known as H218, AGR16, or S1P2, EDG6 also known as S1P4 and EDG8 also known as S1P5) to elicit biological responses. These receptors are coupled differentially via G(i), G(q), G(12/13) and Rho to multiple effector systems, including adenylate cyclase, phospholipases C and D, extracellular-signal-regulated kinase, c-Jun N-terminal kinase, p38 mitogen-activated protein kinase and non-receptor tyrosine kinases. These signaling pathways are linked to transcription factor activation, cytoskeletal proteins, adhesion molecule expression, caspase activities, etc. Therefore sphingosine 1-phosphate can affect diverse biological responses, including mitogenesis, differentiation, migration and apoptosis, via receptor-dependent mechanisms. Additionally, S1P has been proposed to play an intracellular role, for example in Ca(2+) mobilization. Recently, it was shown (Keller et al., 2014 Nature Communications, 5, 5215) that S1P plays an important role in bone-formation in adults through S1P3 signaling.
The 2nd loop of the 7 transmembranal receptor S1P3, also named EDG3 was shown to be involved in angiogenesis. Licht et al. 2003 (Blood, 102, 2099-2107) represented induction of pro-angiogenic signaling, and synergism with known angiogenic factors, by a nine-amino acid peptide having the sequence Myristyl-Gly-Met-Arg-Pro-Tyr-Asp-Ala-Asn-Lys-Arg (SEQ ID NO: 1) derived from the C-terminal end of the 2nd loop of the EDG3 (residues 143-151). No shorter sequences are disclosed in this publication.
WO 01/81408 and its US counterpart, U.S. Pat. No. 6,864,229, disclose peptides and peptide conjugates derived from the third intracellular loop of certain G-protein coupled receptors (protease-activated receptor-1 (PAR1), PAR2, PAR4, CCKB, CCKA, SSTR2 and MC4). These documents do not provides sequence and/or activity of peptides derived from the second loop of G-protein coupled receptors, nor they disclose or suggest peptides or conjugates derived from the S1P3/EDG3 G-protein coupled receptor.
WO 2004/022576 suggests peptides and analogs, of at least five amino acids, derived from the 2nd loop of the 7 transmembranal receptor EDG3, useful in stimulation of angiogenesis. Disclosed peptides contain at least 6 amino acids of the native sequence, or are analogs of these peptides, for example the compound Nle-Arg-Pro-Tyr-Asn-Ala (SEQ ID NO:2), derived from the sequence Met-Arg-Pro-Tyr-Asp-Ala (SEQ ID NO:3) of the native EDG3 sequence. Long peptide sequences are not optimal as therapeutic agents due to their flexible conformation, which affects activity and selectivity, and they comprise more cleavage sites for peptidases.
U.S. Pat. No. 6,075,136 related to human prostate-associated serine protease (PRASP) describes an assay for measuring PRASP activity which measures the hydrolysis of the synthetic peptide methyl-O-succinyl-Arg-Pro-Tyr-NH-p-nitroanilide that serves as a universal substrate for chymotrypsin-like serine proteases, including PSA (Christensson, A. et al. 1990, Eur. J. Biochem. 194:755-763).
There is therefore an unmet need for providing improved peptides which are more appropriate as drug candidates for stimulation of angiogenesis in conditions were insufficient blood-supply occurs.